Cytokine Assay Manual

8/14/2019 Cytokine Assay Manual

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Cytokine AssayInstruction Manual

For technical support, call your local Bio-Rad office or

in the US, call 1-800-4BIORAD (1-800-424-6723).

For research use only. Not for diagnostic procedures.

Bio Plex

Precision Pro

TM

-

Requires Bio-Plex

Manager 4.1 software

(or later versions)


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Table of Contents

Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Section 2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Section 3 Required Materials . . . . . . . . . . . . . . . . . . . . .4

Section 4 Recommended Materials . . . . . . . . . . . . . . . . 5

Section 5 Sample Preparation . . . . . . . . . . . . . . . . . . . . 6

Section 6 Standard Preparation . . . . . . . . . . . . . . . . . . . 7

Section 7 Control Preparation (Optional) . . . . . . . . . . . . 9

Section 8 Assay Instructions . . . . . . . . . . . . . . . . . . . . 10

Plan Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Prepare Coupled Magnetic Beads . . . . . . . . . . . . . 11Calibrate Vacuum Apparatus . . . . . . . . . . . . . . . . . 11

Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Section 9 Data Acquisition . . . . . . . . . . . . . . . . . . . . . . 15

Section 10 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . 20

Section 11 Safety Considerations . . . . . . . . . . . . . . . . . .24


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Section 1Introduction

Bio-Plex Precision Pro cytokine assays are highly sensitive magneticbead-based multiplex assays that allow the accurate measurement of low

levels of cytokines in diverse matrices including serum, plasma, and culture

supernatant. The multiplexing feature makes it possible to quantitate the

level of multiple cytokines in a single well of a 96-well microplate in just

3 hr, using as little as 12.5 l of serum or plasma, or 50 l of culture

supernatant.

As one of the most recent additions to the Bio-Plex suspension arraysystem, these assays incorporate magnetic beads into their design. The

magnetic beads allow the use of an assay protocol similar to non-

magnetic Bio-Plex cytokine assays, with the option of using magnetic

separation of wash steps instead of vacuum filtration (and allows

automation of many of the steps). The 25-bead map in Bio-Plex

Manager 4.1 software (or later versions) is required for data acquisition.

These assays are offered in a convenient kit format that includes assay,reagent, and diluent components in a single box. Standard diluents for

serum and plasma are included, as are additional Iylophilized cytokines

which can be used to prepare user-specified quality controls.

For a current listing of Bio-Plex Precision Pro cytokine assays, visit us on

the Web at www.bio-rad.com/bio-plex/

1


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Section 2Principle

TechnologyThe Bio-Plex suspension array system is built around three core

technologies. The first is a novel technology that uses up to 100 unique

fluorescently dyed beads (xMAP technology) that permit the simultaneous

detection of up to 100 different types of molecules in a single well of a

96-well microplate. The second is a flow cytometer with two lasers and

associated optics to measure the different molecules bound to the

surface of the beads. The third is a high-speed digital signal processor

that efficiently manages the fluorescent output.

Assay Format

The principle of these 96-well plate-formatted, bead-based assays is similar

to a capture sandwich immunoassay. An antibody directed against the

desired target cytokine is covalently coupled to internally dyed beads. The

coupled beads are allowed to react with a sample containing the target

cytokine. After a series of washes to remove unbound protein, a

biotinylated detection antibody specific for a different epitope is added to

the reaction. The result is the formation of a sandwich of antibodies around

the target cytokine. Streptavidin-phycoerythrin (streptavidin-PE) is then

added to bind to the biotinylated detection antibodies on the bead surface.

Data Acquisition and Analysis

Data from the reaction are then acquired using the Bio-Plex suspension

array system (or Luminex system), a dual-laser, flow-based microplate

reader system. The contents of the well are drawn up into the reader.

The lasers and associated optics detect the internal fluorescence of the

individual dyed beads as well as the fluorescent signal on the bead

surface. This identifies each assay and reports the level of target protein

in the well. Intensity of fluorescence detected on the beads indicates the

relative quantity of targeted molecules. A high speed-digital processor

efficiently manages the data output, which is further analyzed and

presented as fluorescence intensity on Bio-Plex Manager software, theaccompanying software package.

2


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Assay Workflow

3

Add beads

Wash

Add standards, controls,

and samples, 1hr

Wash

Add detection antibody, 30 min

Wash

Add streptavidin-PE, 10 min

Wash

Resuspend, acquire data

Prewet wells


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Section 3Required Materials

Bio-Plex Precision Pro assays are offered in a convenient kit formatthat includes assay, reagent, and diluent components all in a single box

(does not require separate reagent and diluent kits). These assays require

the use of Bio-Plex Manager software version 4.1 or higher.

Storage and Stability

Kit components should be stored at 4C and should never be frozen.

Coupled magnetic beads and streptavidin-PE should be stored in the

dark. All components are guaranteed for up to 6 months from the date

of purchase when stored as specified in this manual.

Coupled magnetic beads (25x) 1 vial

Detection antibodies (10x) 1 vial

Standard 2 vials

Control 1 vial

Standard diluent (serum) 10 ml

Standard diluent (plasma) 10 ml

Sample diluent 15 ml

Assay buffer 75 ml

Wash buffer 150 ml

Detection antibody diluent 15 ml

Streptavidin-PE (100x) 1 vial

Sterile filter plate (96-well) 1 plate

Sealing tape 1 pack of 4

Component Units

4


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Section 4Recommended Materials

For optimal results, the use of the items below is recommended.

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Pipets and pipet tips, sterile distilled

water, aluminum foil, absorbent

paper towels, 1.5 ml microcentrifugetubes, 15 ml culture tubes


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Section 5Sample Preparation

This section provides instructions for preparing samples derived fromserum, plasma, and culture supernatant. For sample preparations not

mentioned here, consult the publications listed in Bio-Rad bulletin 5297,

available for download at discover.bio-rad.com

Serum and Plasma Samples

Note that for plasma samples, EDTA tubes are recommended; however,

sodium citrate tubes are acceptable. Extremely lipemic samples may be

filtered with a 0.22 m filter to prevent clogging. Hemolyzed samples arenot suitable for Bio-Plex Precision Pro cytokine assays.

1. Collect and process the serum or plasma samples and assay

immediately or freeze at 20C. Avoid repeat freezing and thawing.

2. Centrifuge the samples at 13,200 rpm for 10 min at 4C

to clear the samples of precipitate. Alternatively, carefully filter the

samples with a 0.22 m filter to prevent instrument clogging.

3. Immediately dilute 1 volume of sample with 3 volumes of sample

diluent. Keep the samples on ice until ready for use.

Culture Supernatant Samples

1. Collect and process the culture supernatant samples and assay

immediately or freeze at 20C. Avoid repeat freezing and thawing.

2. If required, dilute the culture supernatant with culture medium.Serum-free culture medium should contain carrier protein (such as

BSA) at a concentration of at least 0.5%. Keep the samples on ice

until ready for use.

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Section 6Standard Preparation

Two tubes of Iyophilized cytokine standard are provided in each Bio-Plex

Precision Pro cytokine assay. However, only one of the tubes is required per

96-well plate. The product insert provided with the assay lists the

concentration of the reconstituted standard. This procedure will prepare

enough standard to run each dilution in duplicate.

Reconstitute Standards

1. Gently tap the glass vial containing the lyophilized cytokine standard

on a solid surface to ensure the pellet is at the bottom.

2. Reconstitute 1 vial of lyophilized standard with 500 l of the

appropriate standard diluent. Do not use assay buffer to dilute

standards.

3. Gently vortex 13 sec and incubate on ice for 30 min. Be

consistent with the incubation time for optimal assay performance.

Prepare Standard Dilution Series

The cytokine concentrations specified for the 8-point standard dilution set

have been selected for optimized curve fitting using the 5-parameter

logistic (5PL) or 4-parameter logistic (4PL) regression in Bio-Plex

Manager software. Results generated using dilution points other than

those listed in this manual have not been optimized.

1. Label a set of 1.5 ml Eppendorf tubes as shown in the diagram on

the next page.

Serum Serum standard diluent

Plasma Plasma standard diluent

Culture supernatant Same culture medium usedto prepare samples

Sample Standard Diluent

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2. Pipet the appropriate volume of standard diluent into the tubes (see

diagram below). Use serum standard diluent for serum samples,

plasma standard diluent for plasma samples, and culture medium for

culture samples.

3. Add 25.6 l of the reconstituted standard to the first 1.5 ml tube

containing 374.4 l of standard diluent. Vortex gently. This is

identified as S1 in the diagram below and in the product insert

provided with assay.

4. Continue making serial dilutions of the standard as shown. After

making each dilution, vortex gently and change the pipet tip after

every transfer.

NOTE: Running an additional two 0 pg/ml blanks is stronglyrecommended. Use 50 l of the appropriate standard diluent as the

blank sample. The 0 pg/ml points should be formatted as blanks,

not as points in the curve, when using Bio-Plex Manager software.

The blank wells are also useful for troubleshooting and determining

LOD.

5. Keep the standards on ice until ready for use. Standards should be

used immediately and should not be frozen for future use.Standard Dilution Series

8


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9

Section 7Control Preparation (Optional)

One tube of lyophilized cytokine control is provided in each Bio-Plex PrecisionPro cytokine assay. The preparation of high, medium, and low controls is

optional to monitor plate-to-plate variations. This section provides instructions

on how to reconstitute the Iyophilized control. The product insert provided with

the assay lists the concentration of the reconstituted control. The reconstituted

control can then be further diluted to prepare any concentration of user-

specified quality controls. To ensure optimal assay performance, the cytokine

controls should be prepared in a manner consistent as that used to prepare thecytokine standards.

Reconstitute Cytokine Controls

1. Gently tap the glass vial containing the lyophilized cytokine control on

a solid surface to ensure the pellet is at the bottom.

2. Reconstitute 1 vial of lyophilized control with 500 l of the appropriate

diluent. Do not use assay buffer to dilute controls. This is identified

as C0 in the product insert provided with the assay.

3. Gently vortex 1 3 sec and incubate on ice for 30 min. Be consistent

with the incubation time to ensure optimal assay performance.

4. The reconstituted cytokine control should be further diluted to create

the desired QC samples in the same diluents specified in the table

above. To obtain the concentration of each reconstituted cytokine

control, refer to C0 in the product insert provided with the assay.

Serum Serum standard diluent

Plasma Plasma standard diluent

Culture supernatant Same culture medium used

to prepare samples

Sample Diluent


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10

Section 8Assay Instructions

The following instructions apply to Bio-Plex Precision Pro cytokineassays. All of the necessary components are provided premixed for ease

of use.

Plan Experiment

1. Assign which wells of a 96-well plate will be used for each standard,

control, and sample (see the example below).

2. Determine the total number of wells that will be used in the assay.Include a 25% excess (or add 2 wells for every 8 wells used) to

ensure that enough diluted coupled beads, detection antibodies,

and streptavidin-PE are prepared.

Example Plate


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Prepare Coupled Magnetic Beads

Protect the beads from light by covering the tubes with aluminum foil.

Keep all tubes on ice until ready to use.

1. Vortex the coupled beads (25x) at medium speed for 1520 sec.

2. Prepare a sufficient volume of coupled beads (1x) using assay

buffer. Each well requires 2 l of coupled beads (25x) adjusted to a

final volume of 50 l with assay buffer (refer to the example below).

Calibrate Vacuum ApparatusThe vacuum apparatus must be calibrated at the beginning of the assay

to ensure an optimal bead yield. For more detailed instructions, refer to

the Bio-Plex suspension array system hardware instruction manual.

1. Prewet all the wells of a 96-well filter plate with 100 l of assay buffer.

2. Place the filter plate on the vacuum apparatus and turn on the

vacuum to the maximum level.

3. Press on the filter plate and note the time required to remove the

buffer from the wells by vacuum filtration. The evacuation time

should be 25 sec.

If the evacuation time is 5 sec, the pressure is too low. Close the

vacuum control valve slightly and repeat steps 13.

# of Wells 25x Beads (l) Assay Buffer (l) Total Volume (l)

96

48

32

24

240

120

80

60

5,760

2,880

1,920

1,440

6,000

3,000

2,000

1,500

Example Bead Calculations


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12

Assay Procedure

Bring all buffers to room temperature. Avoid bubbles when pipetting.

Assay Key The following terms are repeated throughout the assay

procedure. Refer to these detailed instructions when wash, incubate, and

vacuum-filter are shown in bold.

1. Equilibrate the diluted standards, samples, and controls at roomtemperature for 20 min prior to use.

2. Prewet and block the desired number of wells in a 96-well filter plate

with 100 l of assay buffer and vacuum-filter. If fewer than 96 wells

are required, mark the plate to identify the unused wells for later use

and cover the unused wells with sealing tape.

3. Vortex the coupled magnetic beads (1x) for 1520 sec at medium

speed. Add 50 l to each well and immediately vacuum-filter.

4. Wash twice.

5. Gently vortex the diluted standards, controls, and samples for 13

sec. Add 50 l of standard, control, or sample to each well, changing

the pipet tip after every volume transfer. Incubate for 1 hr.

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Gently cover the filter plate with a new sheet of sealing tape. Place

the filter plate on a microplate shaker and then cover with aluminum

foil. Shake the filter plate at room temperature at 1,100 rpm for

30 sec, then at 300 rpm for the specified incubation time.


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13

6. While the samples are incubating, perform a 30 sec quick-spin

centrifugation of the detection antibody (10x) prior to pipetting to

collect the entire volume at the bottom of the vial.

7. Prepare a sufficient volume of detection antibodies (1x) using

detection antibody diluent. Each well requires 2.5 l of detection

antibodies (10x) adjusted to a final volume of 25 l with detection

antibody diluent (refer to the example below).

8. After incubating the samples, slowly remove and discard the sealing

tape, then vacuum-filter.

9. Wash 3 times.

10. Vortex the detection antibodies gently and add 25 l to each well.

Incubate for 30 min.

11. While the detection antibodies are incubating, perform a 30 sec

quick-spin centrifugation of the streptavidin-PE (100x) prior to

pipetting to collect the entire volume at the bottom of the vial.

12. Prepare a sufficient volume of streptavidin-PE (1x) using assay buffer.

Each well requires 0.5 l of streptavidin-PE (100x) adjusted to a final

volume of 50 l with assay buffer (refer to the example on the

following page).

Example Detection Antibody Calculations

# of Wells10x Detection

Antibody (l)Total Volume (l)

96

48

32

24

300

150

100

75

2,700

1,350

900

675

3,000

1,500

1,000

750

Detection Antibody

Diluent (l)


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14

13. After the detection antibody incubation, slowly remove and discard

the sealing tape, then vacuum-filter.

14. Wash 3 times.

15. Vortex the streptavidin-PE (1x) vigorously and add 50 l to each well.

Incubate for 10 min.

16. After the streptavidin-PE incubation, slowly remove and discard the

sealing tape, then vacuum-filter.

17. Wash 3 times.

18. Add 125 l of assay buffer to each well. Incubate for 30 sec to

resuspend the beads. Acquire the data immediately as described in

Section 9.

# of Wells100x

Streptavidin-PE(l)

Total Volume (l)

96

48

32

24

60

30

20

15

5,940

2,970

1,980

1,485

6,000

3,000

2,000

1,500

Assay Buffer (l)

Example Streptavidin-PE Calculations


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Section 9Data Acquisition

Bio-Plex Precision Pro cytokine assays require the use of Bio-PlexManager software version 4.1 or higher. Recommendations for acquiring

data using the Bio-Plex suspension array system are listed below.

Alternatively, refer to the Bio-Plex Manager software user guide or the

instructions provided with the Luminex instrument.

Prepare System

1. Empty the waste bottle and fill the sheath fluid bottle before starting

(if HTF not present). This will prevent fluidic system backup andpotential data loss.

2. Turn on the reader and microplate platform (and HTF if present). Allow

the system to warm up for 30 min.

3. Select Start up and follow the instructions to prepare the reader

to acquire data. If the system is idle for 4 hr, the lasers will automatically

turn off and a 30 min warm-up period will again be required prior toacquiring data. Select Warm up and wait for the optics to reach

operational temperature.

Calibrate With High RP1 Target Value

Calibrate using Bio-Plex calibration beads and target values. Daily

calibration is recommended before acquiring data.

1. Select Calibrate and confirm that the default values for CAL1

and CAL2 are the same as the values on the Bio-Plex calibrationbead labels. Use the Bio-Plex High RP1 target value for CAL2

calibration for Bio-Plex Precision Pro cytokine assays.

NOTE: When acquiring data for Bio-Plex Precision Pro cytokine

assays with a Luminex instrument, Luminex software, and Luminex

calibration beads, it is necessary to convert the Luminex CAL2

calibration bead RP1 target value using the following equation:

Bio-Plex High RP1 target value = (Luminex RP1 target value) x 4.55


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Add the new target value to the Luminex software by selecting

Calibrate, then New under the Reporter Channel in the Start

Calibration dialog. Enter the new target value and save it as a new lot.

Then calibrate using the new RP1 target value.

2. Select OK and follow the instructions for CAL1 and CAL 2 calibration.

Prepare Protocol

1. Open a new protocol by selecting File, then New from the main

menu. Locate the steps at the left of the protocol menu.

NOTE: To minimize data entry, preset lot-specific Bio-Plex Precision

Pro cytokine assay protocols are available for download at

www.bio-rad.com/bio-plex

2. Select Step 1 (Describe Protocol) and enter information about the

assay.

3. Select Step 2 (Select Analytes) and choose the panel for Cytokines.

Choose the target proteins for the assays on the plate. Note that this

information will already be entered with the preset downloaded

protocol.

Plate Formatting Example

16


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4. Select Step 3 (Format Plate) and click on the Plate Formatting tab.

Click on and drag the cursor over all the wells that contain

standards. Then click on and drag the cursor over the wells that

contain blanks. Repeat with to identify all the wells that contain

controls and to identify all the wells that contain samples.

NOTE: If the preset protocol was downloaded, a formatted plate will

already be provided. Make any necessary changes to the preset

formatted plate to match your plate setup.

5. Select Step 4 (Enter Standards Info) to enter standards information.Note that this information will already be entered with the presetdownload protocol.

a) Select each analyte individually from the pull-down cell.

b) Select the Enter Automatically option and then select the mostconcentrated value as S1.

c) Enter the concentration of S1 from the product insert providedwith the assay.

d) Enter the dilution factor as 4 and select Calculate. The standardsinformation will be populated for the selected analyte.

e) Deselect the box for same concentration values for all analytes.Repeat steps 5a through 5d for each analyte in the assay.

6. Select Step 5 (Enter Controls Info) to enter controls information. Thisis where the concentration of the user-specified controls is enteredinto the protocol.

a) Select each analyte individually from the pull down cell.

b) Enter the description, concentration, and dilution information for

each user-specified control.

c) Deselect the box for same concentration values for all analytes.Repeat steps 6a and 6b for each analyte in the assay.

7. Select Step 6 (Enter Sample Info) and enter sample information.


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18

Acquire Data

1. Shake the assay plate at 1,100 rpm for 30 sec immediately before

acquiring data. Failure to do so will result in increased data acquisition

time due to bead settling.

2. Check that the filter plate is flat. While pressing on one end of the

plate, observe the distance that the opposite end of the plate is

raised off a flat surface. If the distance is >1 mm, transfer all contents

to a flat-bottom 96-well plate or another filter plate.

3. Visually inspect the plate and ensure that the assay wells are filled with

buffer prior to placing the plate in the Bio-Plex microplate platform.

4. Slowly remove the sealing tape and any plate cover before placingthe plate in the reader.

5. Select Step 7 (Run Protocol):

a) Specify data acquisition for 100 beads per region.

b) In Advanced Settings, set the Bead Map to 25 region.

NOTE: Bio-Plex Precision Pro cytokine assays contain magnetic

beads and require the use of the 25 region map available in Bio-

Plex Manager software version 4.1 or higher.

c) In Advanced Settings, set the sample size to 50 l.

d) In Advanced Settings, confirm that the default DD gate values

are set to 5000 (low) and 32000 (high).

NOTE: When using a Luminex instrument, set the gates

according to the Luminex procedure located in the manual.

e) Select Start and save the .rbx file. Then follow the instructions for

data acquisition.


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19

6. If acquiring data from more than one plate, empty the waste bottleand refill the sheath bottle after each plate (if HTF not present). SelectWash Between Plates and follow the instructions for fluidicsmaintenance. Then repeat the Prepare Protocol andAcquire Datasteps.

NOTE: Use the Wash Between Plates command after every platerun to reduce the possibility of clogging the instrument.

7. When data acquisition is complete, select Shut Down andfollow the instructions.

Reacquire Data

It is possible to acquire data from a well or plate a second time using the

Rerun/Recovery mode located below Start in Step 7 (Run Protocol).

1. Check the wells where data will be acquired a second time.Any previous data will be overwritten.

2. Remove the buffer by vacuum filtration and add 125 l of assaybuffer to each well. Cover the filter plate with a new sheet ofsealing tape.

3. RepeatAcquire Data steps 16 to acquire data a second time.The data acquired should be similar to the data acquired initially;however, the data acquisition time will be extended since fewerbeads are present in each well.


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Section 10Troubleshooting Guides

This troubleshooting guide addresses problems that may be encountered with

Bio-Plex Precision Pro cytokine assays. If you experience any of the problems

listed below, review the possible causes and solutions provided. This will assist

you in resolving problems directly related to how the assay steps should be

performed. Poor assay performance may also be due to the Bio-Plex array

reader. To eliminate this possibility, we highly recommend use of the Bio-Plex

validation kit. This kit will validate all the key functions of the array reader and

assist the user in determining whether or not the array reader is functioning

properly.

Possible Causes Possible Solutions

20

High Inter-Assay CV

Standards were not

reconstituted consistently

Incubate the reconstituted

standards for 30 min on ice. Always

be consistent with the incubationtime and temperature.

Reconstituted standards and

diluted samples were not stored

properly

Reconstituted standards and diluted

samples should be prepared on ice

as instructed. Prior to plating, the

reconstituted standards and diluted

samples should be equilibrated to

room temperature.

High Intra-Assay CV

Bottom of filter plate not dry Dry the bottom of the filter plate with

absorbent paper towel (preferably

lint-free) to prevent cross-

contamination.


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21


Pipetting technique Pipet carefully and slowly when

adding standards, samples,

detection antibodies, and

streptavidin-PE, especially when

using a multichannel pipet. Use a

calibrated pipet. Change pipet tip

after every volume transfer.

Reagents and assay components

were not equilibrated to room

temperature prior to plating

All reagents and assay components

should be equilibrated to room

temperature prior to plating.

Contamination with wash

buffer during wash steps

During the wash steps, be careful

not to splash wash buffer from one

well to another. Be sure that the

wells are filtered completely and that

no residual volume remains. Also,

be sure that the microplate shaker

setting is not too high. Reduce the

microplate shaker speed to minimize

splashing.

Slow pipeting samples and

reagents across the plate

Sample pipeting across the entire

plate should take less than 4 min.

Reagent pipeting across the entireplate should take less than 1 min.


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22


Low Bead Count

Miscalculation of bead dilution Check your calculations and be

careful to add the correct volumes.

Beads clumped in multiplex

bead stock tube

Vortex for 1520 sec at medium

speed before aliquoting beads.

Vacuum on for too long when

aspirating buffer from wells

Do not apply vacuum to the filter

plate for longer than 10 sec after the

buffer is completely drained from

each well.

Did not shake filter plate enough

before incubation steps and prior

to reading

Shake the filter plate at 1,100 rpm

for 30 sec before incubation steps

and immediately before reading

the plate.

Reader is clogged Refer to the troubleshooting guide

in the Bio-Plex hardwareinstruction manual.

Low Signal or Poor Sensitivity

Standards reconstituted incorrectly Follow the cytokine standard

instructions carefully.

Detection antibody or

streptavidin-PE diluted incorrectly

Check your calculations and be



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23


High Background Signal

Incorrect buffer was used

(for example, assay buffer

used to dilute standards)

Use sample matrix or serum

standard diluent to dilute

cytokine standards.

Spiked 0 pg/ml wells by mistake Be careful when spiking standards.

Do not add any antigens in the 0

(blank) point.

Streptavidin-PE incubated

too long

Follow the procedure incubation

time.

Poor Recovery

Expired Bio-Plex reagents were

used

Check that reagents have not

expired. Use new or unexpired

components.

Incorrect amounts of components

were added

Check your calculations and be


Microplate shaker set to an

incorrect speed

Check the microplate shaker speed

and use the recommended setting.

Setting the speed too high may

cause splashing and contamination.

Use the recommended plate shaker.

Pipetting technique Pipet carefully and slowly when

adding standards, samples,

detection antibodies, and

streptavidin-PE, especially when

using a multichannel pipet. Use a

calibrated pipet. Change pipet tip

after every volume transfer.


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24

Section 11Safety Considerations

Eye protection and gloves are recommended while using this product.

Consult the MSDS for additional information.

Human Source Material. Treat As Potentially Infectious.

The Bio-Plex Precision Pro cytokine assays contain components of

human origin. This material should be handled as if capable of

transmitting infectious agents. Please use universal precautions. The

material has been tested by an FDA approved test and found negative

for HBsAg, HIV 1/2 Ab, HIV-1 Ag, and HCV. No test method can provide

total assurance that hepatitis B virus, hepatitis C virus, human

immunodeficiency virus, or other infectious agents are absent. These

components should be handled at Biosafety Level 2 containment [US

Government publication: Biosafety in Microbiological and Biomedical

Laboratories (CDC, 1999)]. Handle Bio-Plex Precision Pro serum and

plasma standard diluents as potentially biohazardous material under atleast Biosafety Level 2 containment.


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xMAP is a trademark of Luminex Corp.

Costar is a trademark of Coming Costar Corporation. Eppendorf is a trademark of

Eppendorf-Netheler-Hinz GmbH. Luminex 100 and xMAP are trademarks of Luminex

Corporation. Multiscreen is a trademark of Millipore Corporation. Vortex-Genie is a trademark

of Scientific Industries, Inc.

By purchasing this kit, which contains fluorescent labeled microsphere beads authorized by

Luminex, you, the customer, acquire the right under Luminex's patent rights* to use this kit or

any portion of this kit, including without limitation the microsphere beads contained herein, only

with Luminexs laser-based fluorescent analytical test instrumentation known under the name

of Luminex 100, for example as marketed by Bio-Rad Laboratories, Inc. in the Bio-Plex

system.

*Including, but not limited to US patent 5,981,180; 6,046,807; 6,057,107.

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Bio-Rad Laboratories, Inc.

2000 Alfred Nobel Dr.

Hercules, CA 94547 USA

1-800-424-6723 (in the US)

Life ScienceGroup

06-0143 0305 Sig 110610008318 US/EG Rev A

Bio-RadLaboratories, Inc.

Web site www.bio-rad.com USA 800 4BIORAD Australia 02 9914 2800Austria 01 877 89 01 Belgium 09 385 55 11 Brazil55 21 3237 9400Canada 905 712 2771 China 86 21 6426 0808Czech Republic420 241 430 532 Denmark44 52 10 00

Finland09 804 22 00 France 01 47 95 69 65 Germany089 318 84 0Greece 30 210 777 4396 Hong Kong 852 2789 3300

Hungary36 1 455 8800 India 91 124 4029300/5013478 Israel03 963 6050Italy39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460Mexico 55 5200 05 20 The Netherlands 0318 540666New Zealand64 9415 2280 Norway23 38 41 30 Poland48 22 331 99 99Portugal351 21 472 7700 Russia 7 095 721 14 04Singapore 65 6415 3188 South Africa 27 0861 246 723Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland061 717 95 55Taiwan 886 2 2578 7189/2578 7241 United Kingdom 020 8328 2000

Cytokine Assay Manual

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Transcript of Cytokine Assay Manual